Mouse gangliosides

Spiegel, S., Yamada, K.M., Horn, B.E., Moss, J., and Fishman, P.FL (1985) Fluorescent gangliosides as probes for the retention and organization of fibronectin by ganglioside-deficient mouse cells. /. Cell Biol. 100, 721-726. 

Among the gangliosides, GM4 [ct-NeuAc-(2 — 3)-/ -Gal-(l — 0)-Cer] has a relatively simple chemical structure. It has been detected in human and chicken brain and also (119) as a major ganglioside of mouse erythrocytes, chicken-embryonic liver, and egg yolk. With the help of the azidosphingo-sine glycosylation it has been synthesized very efficiently from the neuraminic acid-containing galactosyl donor 11a-/ (Table XI) (120-122). Similarly the thio isomer was obtained from lib-/ and (120,123) the positional isomer from llc-a. 

In contrast to this work on whole brain, Kemp and Stoolmiller138 used cultured mouse-neuroblastoma cells for their study of incorporation of [ lH]2-acetamido-2-deox> -D-mannose into the sialic acid moiety of gangliosides. They were able to demonstrate clearly the precursor-product relationship among CMS, GM2, and GM1. They found that cultured NB41A cells incorporated [3H]2-acetamido-2-deoxy-D-man-nose into the sialic acid moiety of GM3 in less than 10 minutes. Labeled GM2 was not detected in cells incubated for less than 30 minutes, and measurable activity did not appear in G I1 until alter 60 to 90 minutes. These results further supported the concept that the pathway of synthesis of ganglio-tvpe gangliosides proceeds by way of GM3 —> GM2 — GM1. 

Mouse cells transformed by various oncogenic agents had decreased levels of GM1 and more complex gangliosides compared to their normal counterparts (43,45). More recently, it has been reported that transformed hamster cells exposed to butyrate lost many of their transformed properties and acquired a more normal phenotype... 

Column Chromatography. Sepharose beads containing covalently linked gangliosides (0.2 ml packed volume) were placed into a pasteur pipette containing a small amount of glass wool. Columns were washed with HEM containing 50 ug/ml bovine serum albumin (3 ml). Interferon solutions in MEM-albumin (1 ml) were placed on the columns, which were eluted with MEM-albumin at a flow rate of no more than one drop per minute. Fractions of 1 ml were collected and interferon titers determined in each fraction after serial two-fold dilution. Columns onto which mouse fibroblast interferon had been loaded, were eluted with MEM-albumin first, then with 0.07 M N-acetylneuraminyl lactose at pH 2. 

Since interferons appear to be species-specific, we investigated whether the ganglioside fraction from mouse brain was more potent in inhibiting antiviral activity of mouse fibroblast interferon than that obtained from heterologous brain extracts. As seen in Figure 1 bovine brain gangliosides were almost as potent... 

Figure 1. Effects of mouse ( ) and bovine (Q) brain gangliosides on antiviral activity of mouse fibroblast interferon...

Figure 4. Binding of mouse fibroblast interferon to Sepharose-ganglioside col-ums and elution with N-acetylneuraminyl lactose...

Cells were grown in RPMI medium plus 5 % fetal bovine serum in 1 mL total volume as described in Materials and Methods. At 24-hr intervals the cells were counted in a Coulter counter. Control cells 0, cells from cultures containing 1000 lU/mL mouse fibroblast interferon , cells from cultures containing bovine brain gangliosides at a concentration corresponding to 35 p.M sialic acid , cells from cultures containing both interferon (1000 IU/mL) and gangliosides (35 p.M sialic acid). 

Figure 6. Effects of mouse fibroblast interferon L-1210S and L-1210R cells in the presence of increasing ganglioside concentrations...

Under conditions where over 90% of mouse fibroblast interferon was retained (as shown in Figure k) T-cell interferon quantitatively eluted in the breakthrough of the column (Figure 8). T-cell interferon, after passage through the affinity column, was still insensitive to ganglioside inhibition, excluding the possi-... 

Experiment was carried out as described in Figure 1, using 20 lU/mL of both interferons. , Mouse fibroblast interferon , T-cell interferon , T-cell interferon after passage through a Sepharose—ganglioside column (see Figure 8). T EMC titer in the absence of interferon (19). 

Figure 8. Lack of binding of mouse T-cell interferon to Sepharose-ganglioside columns...

Figure 9. Antigrowth activities of mouse fibroblast and T-cell interferons on L-1210S (left) and L1210R cells (right) and the effects of gangliosides on antigrowth activity...

Comparison Of Thymus Gangliosides Of Various Animals. The unique characteristic of thymus ganglioside is its distinct species specificity. We examined thymus of human, calf, rat and mouse in addition to rabbit. As... 

Marcus et al. reported that anti GM1 ganglioside antibody can be used as a T cell marker (11, 12, 13). In collaboration with Tada s group of the University of Tokyo we very recently found that antibody to asialoganglio-side GM1 specifically labels mouse natural killer cells and that the antibody selectively kills the cells in the presence of complement (14). Natural killer cells are known by their specific ability to attack tumor cells. We also have evidence that certain gangliosides are capable of eliciting autoimmune diseases experimentally which we called the ganglioside syndrome (JJ5). For example, some brain gangliosides produce autoimmune lesions in peripheral nerve in susceptible rabbits. This may correspond to the human... 

Germany). Dialyzed upper phase samples of brain and lymphoma cells with from 30,000 to 50,000 cpm were used for each TLC plate. All radiolabeled samples were chromatographed in parellel with mouse brain ganglioside standards extracted as described above. 

Thus we were able to show that partially purified Gj ganglioside preparations (Thy-1 gl) obtained from Thy-1.1 and Thy-1.2 mouse brain and thymus exhibited specific Thy-1 antigenicity (Fig. la). 

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